Composition for the prevention of muscle fatique and skeletal muscle adaptation of strenuous exercise

ABSTRACT

A composition is disclosed suitable for the prevention and/or treatment of muscular energetic deficiencies and states of asthenia for enhancing sport performances and for the treatment of states of heart fatigue, that may take the form of a dietary supplement, dietetic support or of an actual medicine, which comprises as characterizing active ingredients a combination of L-carnitine and/or at least one alkanoyl L-carnitine and creatinol-phosphate.

This application is the U.S. national phase of international applicationPCT/IT00/00308 filed Jul. 21, 2000, which designated the U.S.

The present invention relates to a composition for the prevention andtreatment of muscular energetic deficiencies, states of asthenia andmuscle fatigue, states of heart fatigue and post-infarct conditions andfor enhancing sporting performances.

Accordingly, the composition may take the form and exert the action of adietary supplement or of an actual medicine, depending upon the supportor preventive action, or the strictly therapeutic action, which thecomposition is intended to exert in relation to the particularindividuals it is to be used in.

More particularly the present invention relates to a n orally,parenterally, rectally or transdermally administrable combinationcomposition which comprises as characterizing ingredients:

(a) at least on e carnitine s elected from the group consisting ofL-carnitine, acetyl L-carnitine, propionyl L-carnitine, butyrylL-carnitine, valeryl L-carnitine and isovaleryl L-carnitine or apharmacologically acceptable salt thereof, and

(b) creatinol-phosphate or a pharmacologically acceptable salt thereof.

Compositions comprising carnitines and creatine or phosphocreatine arealready known. WO 98/43499 (Sigma-Tau) discloses a nutritionalsupplement for facilitating the adaptation of skeletal muscle inindividuals undergoing programs of strenuous exercise and counteractingdefatigation and weariness in asthenic individuals, which comprises acombination of L-carnitine, acetyl L-carnitine and propionyl L-carnitineas basic active ingredients which may also comprise creatine and/orphosphocreatine as optionally additive components.

U.S. Pat. No. 4,376,117 (Simes) discloses the magnesium salt ofcreatinol-O-phosphate which is useful in the treatment and prevention ofmyocardiac infarction.

Both carnitine and creatinol-phosphate[1-(2-hydroxyethyl)-1-methylguanidine-O-phosphate] are well known fortheir important metabolic and pharmacological effects which have led toseveral positive pharmacological and clinical findings.

The carnitines are known to play a major role in the processes ofbeta-oxidation of fatty acids in the formation of ATP. They are alsoendowed with important antioxidant activity as demonstrated by theirprotective effect on lipid peroxidation of the cellular phospholipidmembranes and on oxidative stress induced at the myocardial andendothelial cells level. These biochemical effects of the carnitines arereflected by the favourable results obtained in clinical practice withtheir use in the treatment of various forms of atherosclerosis.myocardial ischaemia, peripheral vasculopaties and diabete.

Creatinol-phosphate, which is a compound structurally akin to creatinephosphate, from which it differs in its greater stability and in variousmetabolic and pharmacodynamic aspects, belongs to that group ofphosphagens which play a fundamental role in muscle energy processes. Itis known, in fact, that creatine phosphate is strongly involved in theprocesses responsible, in muscles, for ATP synthesis which is reducedduring muscular exercise.

Creatine, creatine phosphate, creatine phosphokinase, ATP and ADP arefundamental biochemical structures responsible for muscle function,particularly in anaerobic conditions. However, creatine, above allothers, is the essential compound conditioning the remaining steps, itspresence being of fundamental importance for achieving phosphorylationand the associated ATP-related energy processes. Its administrationleads, in fact, to an increase in its muscular concentration and to anincrease in creatine phosphate.

To obtain these effects in human subjects, however, the administrationof high doses of creatine is necessary, up to and beyond 20 g per day,with consequent adverse side effects, particularly at the renal level.Even though only approximately a quarter of the creatine administeredcan be transformed into creatine phosphate, administration of the latteris not a practical proposition on account of its instability and thedifficulty of oral administration. It would, therefore, appear to be ofgreat interest to provide another phosphorous-bearing derivativebelonging to the pool of organic phosphates which is endowed with greatstability and excellent tolerability and can also be administeredorally, such as creatinol-phosphate, the administration of which, evenat low doses, induces a substantial increase in muscular creatine andthe consequent formation of creatine phosphate. Its administrationcauses an increase in muscular strength in human subjects, which is alsomarked in the elderly, as well as the disappearance of asthenia andmuscular weakness in convalescent subjects and the restoration ofcardiac efficiency in subjects who have suffered an infarct.

Potentially even more interesting, however, also with a view to itsaction on muscular activity, are the results of experiments indicatingits ability to stabilise the cell membranes which may be more resistantto attack by reactive oxygen species (ROS).

Since, as is known, one of the effects that forced muscular exercise mayinduce at muscle level are lesions of the muscle fibres themselvesrelated to oxygen toxicity and to the products of lipid peroxidation,one of the favourable effects of creatinol-phosphate consists in itsability to protect the musculature against ROS-induced lesions.

It has now surprisingly been found that a combination compositioncomprising as its characterising components:

(a) at least one carnitine selected from the group consisting ofL-carnitine, acetyl L-carnitine, propionyl L-carnitine, butyrylL-carnitine, valeryl L-carnitine and isovaleryl L-carnitine or apharmacologically acceptable salt thereof, and

(b) creatinol-phosphate or its pharmacologically acceptable salt, isextremely effective for the prevention and treatment of muscular energydeficiencies, states of asthenia and muscle fatigue, states of heartfatigue and postinfarct conditions, and for enhancing sportingperformance, owing to the potent, unexpected synergistic effect exertedby its components.

Toxicology Tests

Both the carnitines and creatinol-phosphate are products known for theirlow toxicity and good tolerability.

In tests performed in rats, doses of L-carnitine and creatinol-phosphatein combination, corresponding to 250 mg/kg of each compound, wereadministered intraperitoneally without the occurrence of any signs oftoxicity. Likewise, no signs of toxicity were detected when 750 mg/kg ofL-carnitine were administered orally in combination with 750 mg/kg ofcreatinol-phosphate. Even prolonged oral administration for one month of200 mg/kg of L-carnitine plus 200 mg/kg of creatinol-phosphate to ratsdid not cause any toxic intolerance reaction. Full blood counts andblood-chemistry tests performed at the end of treatment also failed toreveal any abnormalities worthy of note as compared to controls. Atautopsy, none of the main organs showed any signs of distress.Histological and histochemical investigations confirmed these findings,the results being comparable to those obtained in the control animals.

Muscle Fatigue Test

The method described by Zheng (Zheng R. L., Acta Pharmacol. Sinica, 14,47, 1993) was used for this test in order to observe whether theadministration of L-carnitine or creatinol-phosphate or of the twoproducts in combination might increase reaction time in treated animalsas compared to controls.

In this test, different groups of mice received daily oral doses of 200mg/kg of L-carnitine or 200 mg/kg of creatinol-phosphate or of the twocompounds in combination over the 6-day period preceding the test. Theanimals were immersed in a tank full of water and swimming endurancetime was measured.

Both L-carnitine and creatinol-phosphate increased swimming endurancetime, but the greatest effect was observed in mice treated with theL-carnitine plus creatinol-phosphate combination. In these latteranimals, in fact, swimming endurance time was significantly longercompared to controls, thus confirming the synergistic effect exerted bythe components of the composition (see Table 1).

TABLE 1 Muscle fatigue test Treatment Swimming time (min) Controls 93 ±8 L-carnitine 118 ± 14 Creatinol-phosphate 124 ± 11 L-carnitine +creatinol-phosphate 191 ± 19

Forced Muscular Exercise Test

As is known, forced muscular exercise can produce structural andinflammatory-type lesions at muscle fibre level, due to the increasedoxygen consumption and to production of reactive oxygen species (ROS). Amarker of the level of ROS-induced lipid peroxidation of the muscle maybe obtained from its malondialdehyde (MDA) concentration. For this test,the method described by Husain was used (Husain K., Pathophysiology, 4,69, 1997) as modified by Li (Li J.X., Acta Pharmacologica Sinica, 20,126, 1999) which consists essentially in examining rats on a treadmillat controlled belt speed and preset inclination. In this way the ratswere made to run at a belt speed of 28 m/min and an inclination ofapproximately 5°. Exhaustion of the control animals subjected to theexercise occurred after approximately 90 minutes.

The test was performed on rats receiving daily oral treament over the6-day period preceding the test with 200 mg/kg of L-carnitine or with200 mg/kg of creatinol-phosphate or with the two compounds incombination.

Five minutes and 30 minutes, respectively, after the end of the test,the animals were sacrificed and samples of gastrocnemius muscle weretaken for measurement of the malondialdehyde (MDA) content using thereaction with thiobarbituric acid according to the method described byOhkawa (Ohkawa H. Angl. Biochem 95, 351, 1979).

The results of this test are illustrated in Table 2, which show that thecombination of L-carnitine plus creatinol-phosphate induces a highlysignificant and unexpected reduction in the MDA concentration present inthe muscle samples. This demonstrates the unexpected synergistic effectof L-carnitine and creatinol-phosphate in protecting muscle againstdamages induced by the reactive oxygen species (ROS) produced in thecourse of forced muscular exercise.

TABLE 2 Forced muscular exercise test MDA content in muscle (nmol.g⁻¹)Treatment After 5 min After 30 min Controls 240 ± 4  236 ± 10L-carnitine 218 ± 11 216 ± 14 Creatinol-phosphate 206 ± 16 209 ± 12L-carnitine + creatinol-phosphate 163 ± 14 169 ± 19

Tests of ATP Content in Rabbit Papillary Muscle after Hypoxia

By submitting sections of rabbit papillary muscle to hypoxia a reductionin muscular ATP content can be induced. The presence in perfusion fluidof substances which interact in muscular energy metabolism may limit thehypoxia-induced loss of ATP content in muscle.

For this test a group of New Zealand rabbits were used. The rabbitsreceived intravenous administrations of 100 mg/kg of L-carnitine or 100mg/kg of creatinol-phosphate or the two compounds in combination everyday over the 3-day period preceding the test. After the third day oftreatment, all animals, including control animals, were sacrificed.

After removing the hearts, sections of papillary muscle measuring 1 mmin diameter and 4-5 cm in thickness were isolated. These tissue sectionswere perfused in a thermostatic bath with a 100% O₂-saturated solution.On introducing 100% N₂ into the bath instead of the O₂, hypoxia wasinduced and maintained for the duration of 90 minutes. The tissues werethen maintained in conditions of normal perfusion for a further periodof 90 minutes. The ATP content of the papillary muscle was estimatedaccording to the method described by Strehler (Strehler B.L., Methods inEnzymology III, N.Y. Acad. Press, 871, 1957.

The results of this test are presented in Table 3, which shows that thecombination of L-carnitine plus creatinol-phosphate unexpectedly affordsa surprisingly greater degree of protection against the ATP reduction inpapillary muscle subjected to hypoxia than does L-carnitine andcreatinol-phosphate alone, thus demonstrating the synergistic effect ofthe two compounds present in the composition.

TABLE 3 Test of ATP content of rabbit papillary muscle ATP concentration(mol/g tissue) Treatment Before hypoxia After hypoxia Controls 1.49 ±0.29 0.39 ± 0.046 L-carnitine 1.53 ± 0.15 0.48 ± 0.036Creatinol-phosphate 1.55 ± 0.31 0.68 ± 0.045 L-carnitine +creatinol-phosphate 1.60 ± 0.28 1.18 ± 0.051

Illustrative, non-limiting examples of compositions according to theinvention are reported hereinbelow.

1) L-carnitine 400 mg Creatinol-phosphate 400 mg 2) Acetyl L-carnitine300 mg Creatinol-phosphate 800 mg 3) Carnitine mixture 300 mg(L-carnitine 100 mg, acetyl L-carnitine 100 mg, propionyl L-carnitine100 mg) Creatinol-phosphate 300 mg 4) L-carnitine 200 mgCreatinol-phosphate 200 mg Creatine 100 mg Taurine 50 mg Inosine 50 mgCoenzyme Q₁₀ 25 mg Selenium methionine 50 μg Vitamin E 5 mgBeta-carotene 5 mg 5) L-carnitine 100 mg Creatinol-phosphate 100 mgPhosphocreatinine 100 mg Creatine 100 mg Inosine 100 mg Vitamin C 50 mgVitamin E 5 mg Beta-carotene 5 mg Coenzyme Q₁₀ 25 mg 6) L-carnitine 200mg Creatinol-phosphate 200 mg Fructose-1,6-diphosphate 200 mgMaltodextrin 200 mg Magnesium 10 mg Selenium methionine 50 μg Zinc 10 mgManganese 1 mg Coenzyme Q₁₀ 25 mg 7) L-carnitine 500 mgCreatinol-phosphate 500 mg Creatine 500 mg Coenzyme Q₁₀ 50 mg Vitamin E10 mg Vitamin C 100 mg Beta-carotene 5 mg Magnesium 10 mg

What is meant by pharmacologically acceptable salt of L-carnitine oralkanoyl L-carnitine is any salt of these active ingredients with anacid that does not give rise to unwanted toxic or side effects. Suchsalts are well known to pharmacy experts.

Examples of suitable salts, though not exclusively these, are: chloride;bromide; iodide; aspartate, acid aspartate; citrate, acid citrate;tartrate; phosphate, acid phosphate; fumarate; acid fumarate;glycerophosphate; glucose phosphate; lactate; maleate, acid maleate;orotate; oxalate, acid oxalate; sulphate, acid sulphate,trichloroacetate, trifluoroacetate and methanesulphonate.

A list of FDA-approved pharmacologically acceptable salts is given inInt. J. of Pharm. 33, (1986), 201-217; this publication is incorporatedherein by reference.

What is claimed is:
 1. An orally, parenterally, rectally ortransdermally administrable combination composition which comprises thefollowing components: (a) at least one carnitine selected from the groupconsisting of L-carnitine, acetyl L-carnitine, propionyl L-carnitine,butyryl L-carnitine, valeryl L-carnitine and isovaleryl L-carnitine or apharmacologically acceptable salt thereof, and (b)1-(2-hydroxyethyl)-1-methylguanidine-O-phosphate or a pharmacologicallyacceptable salt thereof.
 2. The composition of claim 1, wherein theweight ratio (a):(b) is from 1:0.1 to 1:1.
 3. The composition of claim1, wherein the pharmacologically acceptable salt of component (a) isselected from the group consisting of: chloride; bromide; iodide;aspartate, acid aspartate; citrate, acid citrate; tartrate; phosphate,acid phosphate; fumarate, acid fumarate; glycerophosphate; glucosephosphate; lactate; maleate, acid maleate; orotate; acid oxalate;sulphate, acid sulphate; trichioroacetate; trifiporoacetate and methanesulphonate.
 4. The composition of claim 1, which further comprisesvitamins, coenzymes, mineral substances, antioxidants, sugars,aminoacids and proteins.
 5. A method of preventing or treating muscularenergetic deficiencies, asthenia, muscle fatigue, heart fatigue,post-infarct heart conditions or enhancing sporting performances, saidmethod comprising orally, parenterally, rectally or transdermallyadministering a combination comprising the following components: (a) atleast one carnitine selected from the group consisting of L-carnitine,acetyl L-carnitine, propionyl L-carnitine, butyryl L-carnitine, valerylL-carnitine and isovaleryl L-carnitine or a pharmacologically acceptablesalt thereof, and (b) 1-(2-hydroxyethyl)-1-methylguanidine-O-phosphateor a pharmacologically acceptable salt thereof.
 6. The method of claim5, wherein the weight ratio (a):(b) is from 1:0.1 to 1:1.
 7. The methodof claim 5, wherein the pharmacologically acceptable salt of component(a) is selected from the group consisting of: chloride; bromide; iodide;an, aspartate, acid aspartate; citrate, acid citrate; tartrate;phosphate, acid phosphate; fumarate, acid fumarate; glycerophosphate;glucose phosphate; lactate; maleate, acid maleate; orotate; acidoxalate; sulphate, acid sulphate; trichioroacetate; trifiporoacetate andmethane sulphonate.